The present invention relates to reagents useful in the quantitative determination of proteolytic enzymes. More particularly the present invention relates to peptide derivatives that are substrates for enzymes of the class E.C.3.4.4. These enzymes cleave amide linkages in peptide chains on the carboxyl side of arginine and lysine residues.
Classical substrates for trypsin, thrombin, and related enzymes have involved amides such as N.sup..alpha. -benzoyl-DL-arginyl-p-nitroanilide, L-lysyl-p-nitroanilide, N.sup..alpha. -benzoyl-DL-arginyl-2-napthylamide and other di, tri, and higher order arginine and lysine peptides with chromogenic amide leaving groups [B. F. Erlanger, et al., Arch. Biochem. Biophys. 95 (1961) 271; A. Riedel and E. Wunsch, Z. Physiol. Chem. 316 (1961) 1959; R. E. Plapinger, et al., J. Org. Chem. 30 (1965) 1781; L. Svendsen, et al.]. The advantage of extending the amino terminal end of either arginyl or lysyl-p-nitroanilide substrates results in improved behavior [L. Svendsen et al., Thrombosis Res. 1 (1972) 267-78, U.S. Pat. No. 3,884,896].
In particularly U.S. Pat. No. 4,070,245 describes compounds of the general structure: EQU A.sub.1 -Glycyl-prolyl-A.sub.2 -NH-A.sub.3
wherein A.sub.1 represents hydrogen or a blocking acyl or sulfonyl group, A.sub.2 represents arginyl or lysyl, and A.sub.3 represents an aromatic hydrocarbon group which may carry substituents. More particularly the above Patent discloses the structure: EQU Glycyl-prolyl-arginyl-p-nitroanilide EQU (abbreviated: Gly-pro-arg-pNA)
The above prior art compound is a tripeptide with a N-terminal glycyl and has a turnover rate for the determination of thrombin, which is higher than the turnover rates of the present invention compounds.
This high turnover rate for Gly-pro-arg-pNA results in the compound not being suitable for the determination of thrombin in an automated system performing a plurality of assays over an extended period of time.
U.S. patent application Ser. No. 970,767, filed 12/18/78, describes a homologous compound such as sarcosyl-prolyl-arginyl-p-nitroanilide.
The present invention compounds illustrated typically by the following structures: EQU .beta.-Alanyl-L-prolyl-L-arginyl-p-nitroanilide EQU (abbreviated: .beta.-Ala-pro-arg-pNA)
and EQU .gamma.-Aminobutyryl-L-prolyl-L-arginyl-p-nitroanilide EQU (abbreviated: Abu-pro-arg-pNA)
are tripeptides preferably with N-terminal .beta.-alanyl or .gamma.-aminobutyryl or derivatives thereof. The invention compounds have unexpectedly suitable turnover rates as substrates for the determination of thrombin and can be used for performance of a plurality of individual assays over an extended period of time, particularly in an automated system.
A number of automated systems have been devised for analyzing the concentration of a particular substance such as thrombin in a chemical specimen such as blood. For example, U.S. Pat. No. 3,748,044 describes a multiple assay system.
In this system assays for thrombin are performed over periods of ten minutes or more. The present invention substrates show linearity of enzyme reaction during the entire time period and permit suitable monitoring and evaluation of all the assays. Prior art substrates that have high turnover rates and do not show such linearity pose a problem in being too highly reactive with thrombin. As is well known in the chemical arts, it is essential for obtaining reliable data that enzymatic assays be performed and evaluated within a period when the rate of the reaction is linear with time [Biochemical Calculations, I. H. Segel, 2nd ed: J. Wiley and Sons Pub. (1976)].
The problem encountered in the use of substrates with high turnover rates may be resolved in part by using very high concentrations of the substrates or very low levels of plasma in the reaction. These are not practical resolutions for the problem in that the use of high substrate concentrations would be very costly, solubility of such substrates in aqueous medium and possible substrate inhibition would present added difficulties, and low levels of plasma would lead to incomplete activation of the factors involved in thrombin generation. The more acceptable resolution for the problem is the employment of substrates with lower turnover rates. The present invention compounds unexpectedly satisfy this need for substrates with lower turnover rates.